Methods for regulating complement cascade proteins using astrovirus coat protein and derivatives thereof

ABSTRACT

The present invention provides a method for modulating the complement cascade by depleting the plasma of the functional activity of complement proteins and thereby reducing or eliminating complement-mediated cell lysis. The invention provides a method for the therapeutic use of coat proteins and derivatives thereof from the Astroviradae family of viruses in the treatment of complement-mediated cell lysis and peptide mediators of inflammation. The invention provides a method for the therapeutic use of coat proteins and derivatives thereof from the Astroviradae family of viruses in the treatment of complement-mediated diseases. Methods are described herein where complement cascade, triggered by either the classical or alternative complement pathways, is prevented from effecting cell lysis and inflammation due to inhibition or depletion of one or more complement components in the serum following administration of astrovirus coat proteins or derivatives.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/304,717filed Nov. 18, 2009 which claims priority to §371 of PCT/US07/12617filed May 25, 2007 which claims priority to U.S. 60/813,685 filed Jun.15, 2006.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under NIH Grant 1R21 AI608734 awarded by the National Institutes of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The invention relates generally to the field of therapeutic interventionin inflammatory and autoimmune disease. More specifically, the inventionrelates to prevention and treatment of complement-mediated tissuedamage, and to viral illness relating to astrovirus infection.

BACKGROUND OF THE INVENTION

Astroviruses are small, non-enveloped icosahedral viruses with asingle-stranded, messenger-sense RNA genome and are known to infect bothmammals and birds. They are estimated to cause 2-17% of children'sdiarrheal illness worldwide. Astroviral infection can be especiallydevastating for children with malnutrition, intestinal parasites, orboth. Even in developed western countries human astrovirus (HAstV)causes a significant economic loss due to parents taking time off fromwork to care for sick children. This trend of economic loss is likely toworsen as increasing numbers of mothers enter the workforce. Preventionor treatment of astrovirus infection in children would have asignificant economic impact on physician and emergency department visitsand lost workdays. In poultry farming, turkey astrovirus has a majoreconomic impact on the turkey farming industry. In particular, turkeyastroviruses cause a rapidly fatal viremic sepsis in young turkeyssuggestive of overwhelming immunologic cascade that likely involves, andmay be driven by, the complement system. Veterinary therapeuticsdesigned to prevent or mitigate the damage of turkey astrovirus would bea significant development for this industry.

There is a great need for complement inhibitors. Currently, noanti-complement therapies are approved for use in humans, despite theknown morbidity and mortality associated with complement disregulationin many disease processes, including such autoimmune diseases assystemic lupus erythematosus, myasthenia gravis, and multiple sclerosis.The impact of complement-mediated tissue injury in such a diverse arrayof diseases has driven the development of many complement inhibitorswith an estimated market of between $2-4 billion annually. For a reviewon complement therapeutics as of 2003, please see the review article byB. P. Morgan and C. L. Harris entitled, “Complement therapeutics;history and current progress” (B. P Morgan and C. L. Harris, 2003.Molec. Immunol. 40, 159-170). The astrovirus coat protein appears tohave extremely strong effects on the complement system, suggesting thatthe ‘active’ portion of the protein may have clinical utility indecreasing tissue damage from complement-mediated diseases. There arecurrently no commercially available anti-complement specificimmunomodulators. There is some evidence that IVIg (intravenousimmuneglobulin) in high doses has anti-complement effects that mayexplain its utility in some autoimmune diseases. IVIg is extremelyexpensive and has safety concerns because it is derived from the bloodof hundreds of donors.

Current candidate compounds for anti-complement therapeutics have thesignificant disadvantage of acting too broadly, or in some cases are notviable due to toxicity. For example, the most powerful anti-complementsubstance known to date, cobra venom factor (CVF), is capable ofvirtually depleting all C3 in the plasma by acting as a stable C3convertase (C3bBb). However, CVF is essentially untenable as a therapydue to the uncontrolled complement activation that results in aprolonged period of decomplementation and vulnerability to overwhelminginfection in some experimental models (Younger, J. G. et al., 2001. J.Appl. Physiol. 90, 2289-2295). Antibody response to the CVF would likelyalso make the therapeutic benefit of this compound too short-lived to beultimately useful in the treatment of chronic disease. The idealanti-complement therapeutic method would be as effective in complementdepletion as CVF but less toxic and less antigenic when administered tothe host. Astrovirus coat proteins and derivatives thereof are capableof regulating complement cascade proteins to an extent comparable withCVF, and thereby are useful for treatment or prevention ofcomplement-mediated tissue damage and mitigation of complement relateddiseases.

SUMMARY OF THE INVENTION

The invention relates to a method for regulating complement cascadeproteins using Astroviridae family viral coat proteins and derivativesthereof, including viral coat subunits, polypeptides, peptides, fusionproteins, and chimeric derivatives of the coat protein. In one aspect ofthe invention, the astrovirus coat protein or derivative is used toinhibit the lytic process of the classical complement pathway byregulating classical pathway proteins. In another aspect of theinvention, the coat protein or derivative is used to inhibit the lyticprocess triggered the alternative complement pathway by regulatingalternative complement pathway proteins. In these aspects of theinvention, the coat protein or derivative prevents the complementcascade from progressing through the terminal pathway of peptide complexformation. A further embodiment of this invention includes inhibitingthe formation of complement pathway components such as C3 convertase,C3b, C5a, C5b, or the complex of peptides known in the art as theMembrane Attack Complex, or MAC. A person of skill in the art willrecognize that testing the inhibition of the complement pathway may beachieved through well-known standard assays. In one embodiment of theinvention, the assay involves using normal human serum to test lysis ofsensitized red blood cells. In another embodiment of the invention, theassay involves using Factor B depleted serum in order to confine theresults of the assay to testing for inhibition of the classical pathway.In a preferred embodiment of this invention, the astrovirus coat proteinor derivative thereof binds to a component of the complement peptide C1.

In other embodiments of the invention, the method provides for anon-infectious virus-like particle (VLP) to regulate the activity ofcomplement proteins. In one embodiment, multiple copies of theastrovirus coat protein or derivatives thereof are expressed on theouter surface of the particle, typically through recombinant expressionof the viral coat protein in the absence of other non-structural viralgenes. In another embodiment, the VLP is produced from another virus,for example Flock House virus, which displays the complement-regulatingportion of the astrovirus coat protein on its surface. The methodfurther discloses the use of peptides derived from astrovirus coatproteins for the regulation of complement and treatment ofcomplement-related disease.

Another aspect of the invention relates to the purification of wildtypeastrovirus virions or coat proteins thereof for use in regulatingcomplement cascade proteins. Using standard viral purificationtechniques, wildtype astrovirus particles may be purified and used forfurther analysis and testing for complement regulating, inhibiting, ordepleting ability.

Another aspect of the invention relates to the recombinant production ofastrovirus coat protein or derivatives. A person of ordinary skill willrecognize that there are many options for the production of recombinantproteins, and these methods may be adapted without undue experimentationfor the purpose of producing large quantities of viral coat proteins. Ina preferred embodiment of the invention, the recombinant astrovirusprotein is produced in a baculovirus system. In another embodiment, therecombinant protein is produced in E. coli. In still furtherembodiments, the proteins are produced in yeast cells. In each of thesecases, recombinant coat proteins thus produced are harvested from theproducer cells and purified by standard protein techniques. A skilledartisan will appreciate that a wide range of eukaryotic expressionsystems, including mammalian cells, is available for recombinantproduction of proteins. A suggested reference for recombinant moleculartechniques is Sambrook et al., Molecular Cloning: A Laboratory Manual,3^(rd) Ed., Cold Spring Harbor Laboratory Press (2001), herebyincorporated by reference. Similar references are well known to those inthe art and readily available for explanations of routine recombinantmolecular biology protocols.

Further embodiments of the invention include the production ofrecombinant proteins that include regions of a second protein fused tothe astroviral coat protein or derivative. Such a fusion or chimericprotein may be used to regulate complement and decreasecomplement-related tissue damage at a specific target site in therecipient by linking the coat protein to an antibody or antibodyfragment.

In another embodiment, the invention is further directed to the use ofastrovirus coat protein or derivatives thereof to treatcomplement-mediated tissue damage and disease. Complement-mediatedtissue damage is frequently associated with autoimmune and otherdiseases with inflammatory pathologies. Astrovirus coat protein orderivatives may be useful in the treatment of rheumatoid arthritis,systemic lupus erythematosus, multiple sclerosis, myasthenia gravis,hemolytic anemia, membranoproliferative glomerulonephritis, serumsickness, Adult Respiratory Distress Syndrome, ischemia-reperfusioninjury (for example, stroke or myocardial infarction), allo- orxeno-transplantation (including hyperacute rejection and Graft VersusHost Disease), Alzheimer's Disease, burn injuries, hemodialysis damage,cardiobypass damage, Paroxysmal Nocturnal Hemoglobinuria, and otherdiseases associated with complement-mediated tissue damage. Further usesinclude veterinary application to treat animal diseases, such as turkeyastrovirus infection.

In another embodiment, the invention is directed towards methods forisolating and purifying astrovirus coat protein by producing coatprotein or derivatives thereof or VLPs to generate a vaccine againstastrovirus infection in humans or animals.

The present invention provides pharmaceutical compositions comprising atleast one astrovirus coat protein or derivatives thereof, and one ormore pharmaceutically acceptable carriers, diluents, or excipients. Inone embodiment, the composition comprises a therapeutically effectiveamount of the astrovirus coat protein or derivatives thereof. In anotherembodiment, the composition comprises at least one other activeingredient effective in treating at least one disease associated withcomplement-mediated tissue damage.

The present invention also provides a method of preventing or treating adisease associated with complement-mediated tissue damage comprisingadministering the pharmaceutical compositions of the present inventionto an animal in need thereof. In one embodiment, the pharmaceuticalcomposition of the present invention is administered as the sole activepharmaceutical agent. In another embodiment, it is used in combinationwith one or more additional therapeutic or prophylactic agent that iseffective for preventing or treating the disease in question. In thisaspect, the method of the present invention comprises administrating thepharmaceutical composition of the present invention before,concurrently, and/or after, one or more additional therapeutic orprophylactic agent effective in treating at least one disease associatedwith complement-mediated tissue damage.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting.

Other features and advantages of the invention will be apparent from thedetailed description, drawings, and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

Table 1 is a listing of astrovirus family members with fully sequencedcoat protein genes. Samples in bold indicate coat protein sequencesidentified in this study; both a unique identifier and the GenBankaccession number are provided for each isolate. Abbreviations: HAstV,human astrovirus; FAstV, feline astrovirus; MAstV, mink astrovirus;PAstV, pocine astrovirus; OAstV, ovine astrovirus; ANV, avian nephritisvirus; TAstV, turkey astrovirus.

Table 2 summarizes the results of RBC lysis assays on HAstV serotypes1-4.

FIG. 1 is an overview of the three pathways of complement activation.The main protein factors and their effector functions are indicated.

FIG. 2 is a graph depicting the results of the RBC assay on HAstV-1 coatprotein. (n=3).

FIG. 3 is a graph depicting the results of the RBC assay on HAstV-1virions. (n=5).

FIG. 4 is a graph depicting the results of the RBC assay on uninfectedcell culture medium. (n=3).

FIG. 5 is a graph depicting the suppressive activity of HAstV-1 virionson RBC lysis.

FIG. 6 is a graph illustrating the comparative time course of RBC lysisof CVF and HAstV-1. (n=3 to 5).

FIG. 7 is a graph depicting the results of the RBC assay used to testinhibition of the classical complement activation pathway by HAstV-1virions and CVF. (n=3).

FIG. 8 is a graph depicting the results of the RBC assay used to testinhibition of the classical complement activation pathway by HAstV-1coat protein compared to NHS (n=3).

FIG. 9 is a graph depicting the results of the RBC assay used to testinhibition of the alternative complement activation pathway by HAstV-1virions, uninfected cell culture medium and CVF. (n=3 to 5).

FIG. 10 is a graph depicting the results of the RBC assay used to testinhibition of the alternative complement activation pathway by HAstV-1virions (n=3).

FIG. 11A is an image of the 7.5% SDS-PAGE gel loaded with C1q, C1r, andC1s, and run in non-reducing conditions with Coomassie blue staining.

FIG. 11B is an image of a nitrocellulose blot transferred from a 7.5%SDS-PAGE gel loaded with C1q, C1r, and C1s, run in non-reducingconditions and probed with wildtype HAstV-1 coat protein.

FIG. 12A-E are collectively a series of illustrative panels indicatingthat HAstV-1 coat protein binds complement protein C1q.

FIG. 12A is an image of an immunoblot using partially purifiedcomplement factor C1 and purified complement factors C2, C3, and C4loaded into a non-reducing 7.5% SDS-PAGE gel (without boiling),electrophoresed, transferred to nitrocellulose, probed with HAstV-1 coatprotein, and incubated with a primary antibody against HAstV-1 virionsand an HRP-conjugated secondary antibody.

FIG. 12B is an image of the same immunoblotting procedure in FIG. 12Awithout using the HAstV-1 coat protein probe.

FIG. 12C is an image of an immunoblot using BSA, C1, C1q, C1r, and C1sloaded into a reducing buffer, boiled, electrophoresed on a 12% SDS-PAGEgel, and subsequently incubated with HAstV-1 coat protein and visualizedas in FIG. 12A.

FIG. 12D is an image of the same blot depicted in FIG. 12C afterstripping and re-blotting with polyclonal antibodies to C1q, C1r, andC1s.

FIG. 12E is an image of a duplicate blot of the experiment in FIG. 12Cwithout using the HAstV-1 coat protein probe.

FIG. 13A-E are images of SDS-PAGE gels (A, C, and E) and immunoblots (Band D) from experiments analyzing HAstV-1 coat protein purificationprocedure and demonstrating the spontaneous oligomerization by the coatprotein.

FIG. 14A is an image of an immunoblot showing a comparison of iC3bproduction in NHS alone, NHS plus HAstV-1 coat protein or NHS plus CVF.

FIG. 14B is a graph depicting the results of an ELISA quantifying theamount of iC3b (in ng/mL) produced in NHS alone, NHS plus HAstV-1 coatprotein or NHS plus CVF.

FIG. 14C is a graph depicting the results of an ELISA quantifying theamount of SC5b-9 (in ng/mL) produced in NHS alone, NHS plus HAstV-1 coatprotein or NHS plus CVF.

FIG. 15A is a graph depicting the reversal of HAstV-1 coat proteininhibition of complement lysis by the addition of exogenous C1 proteinin an RBC lysis assay.

FIG. 15B is a graph depicting the results of an ELISA for quantifying C3(in ng/mL) deposition on zymosan in samples with NHS alone, NHS plusHAstV-1 coat protein (with or without the addition of exogenous C1), orNHS plus CVF (with or without the addition of exogenous C1).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for regulating complement cascadeproteins through the use of coat protein and derivatives thereof fromthe viral Astroviridae family. This invention has multiple uses andadvantages. Astrovirus coat proteins may be used to treatcomplement-mediated diseases or reduce complement-mediated tissue damagein a variety of pathologies in both humans and animals.

The Astrovirus Coat Protein

For a review of the current knowledge concerning astrovirus capsidbiology, please refer to the review entitled “Identification ofstructural domains involved in astrovirus capsid biology” (ViralImmunology 18(1): 17-26, 2005), incorporated herein by reference in itsentirety. Briefly, the Astroviridae constitute a family ofnon-enveloped, icosahedral viruses with a single-stranded,messenger-sense RNA genome. These viruses infect mammals and birds andare a significant cause of gastroenteritis in young children as well asdisease in other animals and avian species, including a fatal viremicsepsis in turkeys. The invention herein discloses, for example, a methodfor an in vitro assembly system in which large quantities of the coatprotein (and coat protein deletion mutants) can be purified using arecombinant baculovirus expression system in insect cells. Otherrecombinant techniques are also contemplated for the production of coatprotein or derivatives. Infectious astrovirus, such as HAstV-1 or othervirions produced in mammalian tissue culture, may also be used.

The Complement System

The complement system comprises a group of related plasma proteins that,when activated, generates an extremely destructive immunologic cascade.The complement system combats infection by a wide variety of methodsincluding lysis of bacteria and infected cells by pore formation (i.e.,formation of the Membrane Attack Complex or “MAC”), opsonization(immune-tagging) leading to ingestion and destruction by white bloodcells, activation of white blood cells, directing white blood cells tothe site of infection, stimulating B-lymphocyte responses, and antibodygeneration. The complement system is activated by three known pathways:the classical pathway, the alternative pathway, and the mannan-bindinglectin pathway (see FIG. 1). Each results in a cascade ofprotein-protein reactions amplifying in an exponential manner thatculminate in an extremely robust immune response. While complement is avital host defense against pathogenic organisms such as bacteria andsome enveloped viruses, its unchecked activation can cause devastatinghost cell damage. Host tissue damage mediated by complement has beenimplicated in a wide variety of diseases including autoimmunepathologies such as: rheumatoid arthritis, systemic lupus erythematosus,multiple sclerosis, myasthenia gravis, autoimmune hemolytic anemia,membranoproliferative glomerulonephritis, and serum sickness. It hasalso been identified as contributing to the pathogenesis of thefollowing diseases: Adult Respiratory Distress Syndrome (ARDS), stroke(ischemia—reperfusion injury), myocardial infarction(ischemia—reperfusion injury), allo- and xenotransplantation (hyperacuterejection & graft versus host disease), Alzheimer's disease, burninjuries, hemodialysis damage, cardiopulmonary bypass damage, andParoxysmal Nocturnal Hemoglobinuria.

Accordingly, the present invention relates to using coat proteins orderivatives thereof from the Astroviridae family of viruses to mitigatethe tissue damage associated with complement cascade proteins. Althoughnot intending to be bound by any particular mechanistic theory, the coatproteins may deplete or inhibit either the classical complement pathway,typically initiated by antibody binding to an antigen followed by the C1protein and fragments thereof, or the alternative pathway, typicallyinitiated by the C3 protein and fragments thereof.

As used herein, the term “astrovirus” refers to any member of theAstroviridae family, including but not limited to mammalian astrovirusspecies such as bovine, feline, human, ovine, porcine, and minkastrovirus, or avian species including chicken, turkey, and duckastrovirus (Table 1).

TABLE 1 Astrovirus family members with fully sequenced coat proteingenes Sequence Length Identifier^(a) Host/Serotype^(b) Nucleotide Aminoacid Location L23513 HAstV-1 2364 788 UK S68561 HAstV-1 2361 787 UKNC_001943 HAstV-1 2361 787 UK AY720892 HAstV-1 2364 788 Germany001-EF138823 HAstV-1 2364 788 CA, USA 002-EF138824 HAstV-1 2370 790 OH,USA 003-EF138825 HAstV-1 2364 788 OH, USA 004-EF138826 HAstV-1 2364 788OH, USA L06802 HAstV-2 2391 797 UK 005-EF138827 HAstV-2 2400 800 CA, USAAF141381 HAstV-3 2385 795 Germany AF117209 HAstV-3 2385 795 USA006-EF138828 HAstV-3 2385 795 CA, USA 007-EF138829 HAstV-3 2385 795 OH,USA DQ070852 HAstV-4 2316 772 Brazil AB025801 HAstV-4 2316 772 JapanAB025802 HAstV-4 2316 772 Japan AB025803 HAstV-4 2316 772 Japan AB025804HAstV-4 2316 772 Japan AB025805 HAstV-4 2316 772 Japan AB025806 HAstV-42316 772 Japan AB025807 HAstV-4 2316 772 Japan AB025808 HAstV-4 2316 772Japan AB025809 HAstV-4 2316 772 Japan AB025810 HAstV-4 2316 772 JapanAB025811 HAstV-4 2316 772 Japan AB025812 HAstV-4 2316 772 Japan AY720891HAstV-4 2316 772 Germany DQ344027 HAstV-4 2316 772 China Z33883 HAstV-42316 772 UK DQ028633 HAstV-5 2352 784 Brazil AB037273 HAstV-5 2352 784Japan AB037274 HAstV-5 2352 784 Japan U15136 HAstV-5 2352 784 UKAB013618 HAstV-6 2337 779 Japan AB031030 HAstV-6 2337 779 Japan AB031031HAstV-6 2337 779 Japan Z46658 HAstV-6 2337 779 UK Y08632 HAstV-7 2376792 Norway AF248738 HAstV-7 2376 792 South Africa Z66541 HAstV-8 2349783 UK AF260508 HAstV-8 2349 783 Mexico 008-EF138830 HAstV-8 2349 783OH, USA 009-EF138831 HAstV-8 2349 783 OH, USA AF056197 FAstV 2451 817 UKNC_004579 MAstV 2328 776 Sweden AB037272 PAstV 2331 777 Japan Y15938PAstV 2352 784 Japan NC_002469 OAstV 2289 763 Scotland NC_003790 ANV2052 684 Japan AB046864 ANV-2 2040 680 Japan AY769615 TAstV-2 2166 722USA NC_005790 TAstV-2 2175 725 USA AY769616 TAstV-3 2175 725 USANC_002470 TAstV 2016 672 USA

The term “coat protein” refers to components of the astrovirus capsid,including but not limited to intact or assembled astrovirus protein coator subunits thereof, precursor proteins, epitopes, monomers, dimers,trimers, oligomers, polypeptides, or peptides.

The term “derivative” refers to components of the astrovirus coat,either purified from wildtype virus or recombinantly produced, which arepartial regions or modifications of the astrovirus coat protein such asisolated coat subunits, truncation or deletion mutants, substitutionmutants, chimeric proteins, fusion proteins, or other proteinscontaining elements of the astrovirus coat in whole or part, in whichthe derivative is capable of regulating complement cascade proteinactivity.

The terms “complement cascade” and “complement protein” refer tocomplement proteins C1-C9 and subunits thereof, including but notlimited to C1q, C1r, C1s, C4a, C4b, C2a, C2b, C3a, C3b, Factor B, Ba,Bb, C3 convertase, C5 convertase, D, Properdin, C5a, C5b, C6, C7, C8,C9, or combinations thereof in the classical, alternative, ormannan-binding lectin complement pathway as would be appreciated by oneof skill in the art.

The term “inhibition” refers to the reduction in the biological functionof an enzyme, protein, peptide, factor, byproduct, or derivative thereofeither individually or in complexes; reduction in the quantity of abiological protein, peptide, or derivative thereof whether in vivo or invitro; or interruption of a biological chain of events, cascade, orpathway known to comprise a related series of biological or chemicalreactions. The term inhibition may thus be used, for example, todescribe the reduction of quantity of a single component of thecomplement cascade compared to a control sample, a reduction in the rateor total amount of formation of a component or complex of components, orthe reduction of the overall activity of a complex process or series ofbiological reactions leading to such outcomes as cell lysis, formationof convertase enzymes, formation of complement-derived membrane attackcomplexes, inflammation, or inflammatory disease. In an in vitro assay,the term “inhibition” may refer to the measurable reduction of somebiological or chemical event, but the person of ordinary skill in theart will appreciate that the measurable reduction need not be total tobe “inhibitory.”

Production of Viral Coat Protein

Numerous sources for the production or isolation of astrovirus coatprotein and derivatives thereof are well understood in the art. Examplesof viral coat protein purification techniques include the use of sucrosegradient sedimentation as well as separation columns, which may purifyviral particles based on size exclusion, affinity binding, ion exchange,hydrophobic interaction (HIC), or other means. In some cases, modifiedcoat proteins or derivative proteins may be difficult to purify tohomogeneity using a column. In these circumstances, the artisan ofordinary skill will appreciate that alternative methods of purification,such as electrophoresis followed by elution and recovery from the gel isan alternative means for preparation of purified viral coat proteins.

In another aspect of the invention, it is contemplated that recombinanttechnology may be employed to produce astroviral coat proteins. In apreferred embodiment of this aspect of the invention, a recombinantbaculovirus is used for in vitro viral coat protein production. Sf9,Sf21, or Tini/High5 insect cells are commonly used in this technique togenerate baculovirus stocks, which subsequently are used to infectproducer cells, which in turn generate the recombinant coat protein.Using such a technique or a variation thereof allows for the large-scaleproduction of both wildtype astrovirus coat protein in addition torecombinant mutants, chimeric proteins, fusion proteins, and virus-likeparticles. Provided herein by way of example infra are methods for theproduction and purification of HAstV-1 (Newcastle) full length coatprotein Ac_(—)1-787, deletion mutant Ac_(—)1-415, and deletion mutantAc_(—)416-787. Other variations of coat protein mutants may similarly beproduced and purified in a similar fashion.

The term “virus like particles” (VLP) refers to non-infectious viralparticles or capsids, which may be produced by recombinant means orthrough the use of chimeric technology in conjunction with VLP fromother viruses that display astrovirus coat protein epitopes or regionsin a polyvalent manner on the particle surface.

As used herein, the term “chimeric” or “fusion” coat protein is intendedto include any recombinant protein capable of inhibiting or depletingcomplement cascade factors which includes a constituent polypeptide,component, or region of the astrovirus coat protein in addition to atleast one region from a second polypeptide or protein, such as anantibody or antibody fragment.

In another embodiment of the invention, the production of recombinantastrovirus coat proteins or derivatives thereof takes place in E. colicells using standard recombinant genetic techniques as will beappreciated by one of skill in art. Astrovirus coat peptides,polypeptides, regions, or whole proteins thus produced are thensubsequently purified on a sucrose gradient, column or similar apparatusas discussed supra. It will be recognized by one of ordinary skill inthe art that production of recombinant proteins is readily adaptable toother systems, such as yeast cells, which likewise involves theintroduction of recombinant DNA into host cells followed by propagation,lysis, and purification of the protein of interest.

Inhibition of Complement-Mediated Lysis by Astrovirus Coat Protein

The invention disclosed herein demonstrates by way of example that thecoat protein of Astroviridae family member HAstV-1 is as effective asCVF in inhibiting complement-mediated cell lysis. Given the structuralsimilarities between members of the Astroviridae family, coat protein orderivatives thereof from other mammalian strains of astrovirus, such ashuman strains HAstV-2 through HAstV-8, bovine, porcine, ovine, feline,mink and poultry strains such as chicken, turkey, and duck astrovirusare contemplated herein as also having anti-complement activity. Table 1provides a non-limiting list of fully sequenced astrovirus familymembers to date.

In order to compare the complement-depleting or inhibiting activity ofHAstV-1 or other astrovirus coat protein to CVF, it is informative totest the astrovirus coat protein in a series of cell lysis experimentsas described in the examples infra. Red blood cells are a sensitive andspecific method for testing serum complement activation by measuring redblood cell lysis. Measurement of the percentage of lysed cells in theassay is therefore a proximal measurement of complement activation inthe experimental sample, typically normal human serum (NHS). The methoddisclosed herein for inhibiting complement mediated lysis is comparablein efficacy to CVF, the strongest complement-depleting substance innature. The inhibition of complement-mediated lysis of RBCs applies notonly to purified coat protein, but also to intact HAstV-1 virions.Accordingly, in another embodiment of the invention, intact astrovirusvirions or virus-like particles are used to deplete complement cascadeproteins from the plasma. Although HAstV-1 virions and purified coatprotein demonstrate similar levels of complement suppression whencompared to CVF, the data presented here demonstrates that astrovirusvirions and coat protein act to suppress serum complement activitythrough an inhibition mechanism as opposed to activation and subsequentdepletion of complement factors exhibited by CVF.

It is important to note that wildtype astrovirus coat protein oftenoligomerizes into trimers (and possibly other higher order oligomers) inthe buffer in which the coat protein is stored. Thus, it is contemplatedthat, in one aspect of the invention, the coat protein in the red bloodcell assays for complement-mediated lysis is a dimer, a trimer, or ahigher-ordered oligomer. The coat protein may precipitate when exposedto calcium ions, an effect which occurs to a lower extent with magnesiumand not at all with EDTA/NaCl. It is contemplated that theseobservations may be indicative of early viral-like particle assembly, asmany icosahedral capsids require calcium ions for assembly. The RBClysis assays described herein contain calcium ions (150 micromolar) andwould therefore be consistent with the observed self-assembly of thecoat protein subunits.

Interactions Between Protein Coat Subunits and Complement Components

An important area of investigation for the optimization of using theinvention disclosed herein is to identify interactions between specificcomplement components and astrovirus coat proteins. This may beachieved, for example, through the use of a modified viral overlay blot,described by way of example infra., which generally involveselectrophoretic gel separation of complement proteins followed bytransfer to a membrane and probing with purified coat protein. Severalcaveats should be noted in this approach. First, the artisan of ordinaryskill will appreciate that while complement proteins C2, C3, and C4 arereadily available as highly purified preparations, the C1 proteincomplex is much more difficult to purify owing to the fact that it is avery labile complex and falls apart easily. As a result, other serumproteins are usually present in C1 preparations. However, C1preparations that are free of other complement factors such as C2, C3,C4, and C5, and routine batch testing with anti-complement antibodies isgenerally sufficient to prevent most cross-contamination with othercomplement factors.

While C1q, a complex molecule, separates into several fragments duringelectrophoresis, C1r and C1s run as homodimers in non-reducing gels.Binding of the astrovirus coat protein in these assays may be specificto homodimers; one of ordinary skill will realize that the same proteinpreparations run under reducing conditions may not reflect binding atall, either because the coat protein is incapable of binding the monomeror because the concentration of the monomer in the preparation is belowdetectable levels.

Formulation and Administration

The present invention provides pharmaceutical compositions comprising atleast one astrovirus coat protein or derivatives thereof, and one ormore pharmaceutically acceptable carriers, diluents, or excipients.Pharmaceutically acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed. Theycan be solid, semi-solid, or liquid. Thus, the pharmaceuticalcompositions of the present invention can be in the form of tablets,pills, powders, lozenges, sachets, cachets, elixirs, suspensions,emulsions, solutions, or syrups.

Some examples of pharmaceutically acceptable carriers, diluents, orexcipients include lactose, dextrose, sucrose, sorbitol, mannitol,starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin,calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone,cellulose, sterile water, syrup, and methyl cellulose. Thepharmaceutical compositions of the present invention can be formulatedso as to provide quick, sustained or delayed release of the activeingredient after administration by employing procedures known in theart.

The pharmaceutical compositions of the present invention are prepared bymixing the astrovirus coat protein or derivatives having the appropriatedegree of purity with pharmaceutically acceptable carriers, diluents, orexcipients. Examples of formulations and methods for preparing suchformulations are well know in the art. See, e.g., Remington'sPharmaceutical Sciences, 18th edition (1995), hereby incorporated byreference.

The pharmaceutical compositions of the present invention are useful as aprophylactic and therapeutic agent for various disorders and diseases asset forth above. In one embodiment, the composition comprises atherapeutically effective amount of the astrovirus coat protein orderivatives thereof. In another embodiment, the composition comprises atleast one other active ingredient effective in treating at least onedisease associated with complement-mediated tissue damage. The term“therapeutically effective amount,” as used herein, refers to the totalamount of each active component that is sufficient to show a meaningfulpatient benefit.

The therapeutically effective amount of the astrovirus coat proteins orderivatives vary depending on such factors as the condition beingtreated, the severity of the condition, the time of administration, theroute of administration, the rate of excretion of the compound employed,the duration of treatment, the co-therapy involved, and the age, gender,weight, and condition of the patient, etc. Determining therapeuticallyeffective amount is well within the skill of a practicing physician.Accordingly, it may be necessary for the therapist to titer the dosageand modify the route of administration as required to obtain the maximaltherapeutic effect.

Under such guidelines, the effective daily dose generally is within therange of from about 0.001 to about 100 milligrams per kilogram of bodyweight, preferably about 0.01-50 mg/kg, more preferably about 0.1-20mg/kg. This can be achieved through a 1-6 times daily dosing regimen.Alternatively, optimal treatment can be achieved through sustainedrelease at a less frequent dosing regimen.

Pharmaceutical formulations may be adapted for administration by anyappropriate route, for example by the oral, nasal, topical (includingbuccal, sublingual, or transdermal), or parenteral (includingsubcutaneous, intracutaneous, intramuscular, intraarticular,intraperitoneal, intrasynovial, intrasternal, intrathecal,intralesional, intravenous, or intradermal injections or infusions)route. For human administration, the formulations preferably meetsterility, pyrogenicity, general safety, and purity as required by FDAOffice and Biologics standards.

Combination Therapies

The present invention also provides a method of preventing or treating adisease associated with complement-mediated tissue damage comprisingadministering the pharmaceutical compositions of the present inventionto an animal in need thereof. While the pharmaceutical compositions ofthe present invention can be administered as the sole activepharmaceutical agent, they can also be used in combination with one ormore additional therapeutic or prophylactic agent that is effective forpreventing or treating the disease in question. In this aspect, themethod of the present invention comprises administrating thepharmaceutical composition of the present invention before,concurrently, and/or after, one or more additional therapeutic orprophylactic agent effective in treating at least one disease associatedwith complement-mediated tissue damage.

For example, the pharmaceutical compositions of the present inventioncan be used to treat rheumatoid arthritis, either alone or incombination with a non-steroidal anti-inflammatory agent (NSAID), acorticosteroid, or a disease modifying anti-rheumatic drug (DMARD).

Examples of NSAID include Salicylates (such as Aspirin, Amoxiprin,Benorilate, Choline magnesium salicylate, Diflunisal, Faislamine, Methylsalicylate, Magnesium Salicylate, and Salicyl salicylate (salsalate)),Arylalkanoic acids (such as Diclofenac, Aceclofenac, Acemetacin,Bromfenac, Etodolac, Indometacin, Ketorolac, Nabumetone, Sulindac, andTolmeti), 2-Arylpropionic acids (such as Ibuprofen, Carprofen, Fenbufen,Fenoprofen, Flurbiprofen, Ketoprofen, Loxoprofen, Naproxen, Tiaprofenicacid, and Suprofen), N-Arylanthranilic acids (such as Mefenamic acid andMeclofenamic acid), Pyrazolidine derivatives (such as Phenylbutazone,Azapropazone, Metamizole, Oxyphenbutazone, and Sulfinprazone), Oxicams(such as Piroxicam, Lornoxicam, Meloxicam, and Tenoxicam), COX-2Inhibitors (such as Etoricoxib, Lumiracoxib, and Parecoxib),Sulphonanilides such as Nimesulide, and others such as Licofelone andOmega-3 Fatty Acids.

Examples of corticosteroid include triamcinolone (Aristocort®),cortisone (Cortone® Acetate Tablets), dexamethasone (Decadron® Elixir),prednisone (Deltasone®), and methylprednisolone (Medrol®),

Examples of DMARD include methotrexate (Rheumatrex®), leflunomide(Arava®), etanercept (Enbrel®), infliximab (Remicade®), adalimumab(Humira®), anakinra (Kineret®), sulfasalazine (Azulfidine EN-Tabs®),antimalarials, gold salts, d-penicillamine, cyclosporin A,cyclophosphamide and azathioprine.

Soliris™ (eculizumab) is a humanized anti-05 monoclonal antibody. It hasbeen approved by the FDA for the treatment of the rare form of hemolyticanemia, paroxysmal nocturnal hemoglobinuria. In one embodiment, thepharmaceutical compositions of the present invention can be used incombination with Soliris™ in treating paroxysmal nocturnalhemoglobinuria, heart disease, pulmonary diseases, autoimmune diseases,asthma, as well as the ancillary care of transplant.

The pharmaceutical compositions of the present invention can beadministered with the additional agent(s) in combination therapy, eitherjointly or separately, or by combining the pharmaceutical compositionsand the additional agent(s) into one composition. Dosage administrationand adjustment is done to achieve maximal management of the conditionsto be treated. For example, both the pharmaceutical compositions and theadditional agent(s) are usually present at dosage levels of betweenabout 10 and about 150%, more preferably, between about 10 and about 80%of the dosage normally administered in a monotherapy regimen.

Hereditary angioedema (HAE) is a very rare genetic disorder caused byreduced levels or non-functional C1-inhibitor. C1-inhibitor naturallyregulates C1 activation and treatment of acute edema in these patientsrequires substantial infusion of C1-inhibitor or plasma transfusion.Because astrovirus coat protein functionally blocks C1 activation, thiswould be a potential therapy and would fill a therapeutic need becauseC1-inhibitor has to be purified from human sera from multiple subjectsand therefore has the potential to contaminated with human bloodbornepathogens. Therapeutic administration of astrovirus coat protein or aderivative thereof may potentially be used to inhibit C1 either inadjunct therapy with C1-inhibitor or as a stand-alone therapeutictreatment.

EXAMPLES

The invention is further illustrated by the following examples. Theexamples are provided for illustrative purposes only. They are not to beconstrued as limiting the scope or content of the invention in any way.

Example 1 Cell Lines and Viruses

For baculovirus production of astrovirus coat protein, SpodopteraFrugiperda cells (line IPLB-Sf21) (Vaughn, J. L. et al., 1977. In vitro.13, 213-217) were propagated in TC100 medium supplemented with 10%heat-inactivated FBS as described previously (Scheneemann, A. et al.,1993. J. Virol. 67, 2756-2763). Virus stocks of the recombinantbaculoviruses encoding the wildtype HAstV-1 coat protein gene anddeletion mutants were prepared by infecting Sf21 cells at a multiplicityof infection (MOI) of 1 in cell growth medium and allowing the infectionto proceed for 5 to 7 days. Following the infection period, cell debriswas removed in a low speed spin and virus contained in the medium wastitered by plaque assay and stored at 4° C.

For propagation of infectious astrovirus particles, CaCo-2 cells (J.Fogh and G. Trempe, 1975. New human tumor cell lines. In: Fogh J (ed)Human tumor cell lines in vitro. Plenum, New York, pp 115-159) werepropagated in minimum essential medium with 10-20% heat-inactivated FBSaccording to instructions (ATCC). A cell-adapted strain of HAstV-1(Oxford) (kindly provided by Dr. D. K. Mitchell, Eastern VirginiaMedical School, Norfolk, Va., USA) was propagated in CaCo-2 cells(Willcocks, M M. et al., 1990. Arch. Virol. 113:73-81). Briefly, cellmonolayers in minimum essential medium lacking FBS were infected with aviral inoculum containing 10 μg/ml of trypsin type IX (Sigma) and viruswas allowed to adhere for 1 h at 37° C. The inoculum was removed andmedium containing 2 μg/ml of trypsin was added; cells were thenincubated for approximately 48 hours at 37° C. Following incubation,viral suspensions were released from the cells by 3 cycles offreeze/thaw. Cell debris was then removed in a low speed spin and thesupernatant containing virus was aliquoted and stored at −80° C.Cell-adapted strains of HAstV types 2-7, previously propagated as above,were provided by Dr. D. K. Mitchell.

Example 2 Real-Time Reverse Transcription PCR

To quantify HAstV stocks, a real-time PCR method was developed. Toisolate total RNA, 400 μL of CaCo-2 cells lysates infected with HAstV-1were diluted 1:5 in 1× minimum essential medium. RNA was then extractedusing Trizol (Invitrogen) per manufacturer's instructions. Followingisolation, RNA was treated with DNAseI (Promega) for 30 min at 37° C.and the enzyme was then inactivated at 65° C. for 10 min. RNA was storedat −80° C.

One-step real-time RT-PCR was performed using the iCycler IQ™ system(Bio-Rad). The real-time RT-PCR reaction was assembled using theSuperscript III Platinum Syber Green® 1-Step qRT-PCR kit (Invitrogen).Briefly, a reaction mixture was made, containing 12.5 μL 2× Syber® GreenRT-PCR Reaction Mix, 0.5 μL each of 10 μM forward primer ORF1a-F1 andreverse primer ORF1a-R1 (targeting a conserved portion of the serineprotease gene of the HAstVs, 200 nM final concentration), 0.5 μL iScriptReverse Transcriptase for One-Step RT PCR, 6.00 μL sterile water and5.00 μL of the total RNA (isolated as described above). cDNA synthesiswas achieved by incubating the reaction for 10 min at 50° C., followedby inactivation of iScript RT at 95° C. for 5 min. PCR cycling anddetection included 45 cycles of incubation at 95° C. for 10 sec, 55° C.for 30 sec, and 72° C. for 30 sec, respectively. For the melt curve,samples were incubated at 95° C. for 1 min, 57° C. for 1 min, followedby 80 cycles of incubation for 10 sec, starting at 57° C., andincreasing at 0.5° C. increments with each successive cycle. To generatea standard curve, an RNA standard was prepared by T7-mediated in vitrotranscription (Ambion) of a genome-length cDNA clone (pAVIC) for HAstV-1(U. Geigenmüller, et al. 1997. J. Virol. 71, 1713-1717.). The RNAstandard was serially diluted from 10¹⁰ to 10⁰ and the standard curvewas established by plotting the threshold cycle vs. log starting copynumber for each dilution. Log starting copy number of the viral RNAcontained in the CaCo-2 cell lysate was then extrapolated from theequation of the standard curve line, Y=mX+b, where Y=threshold cycle(C_(T)), m=slope of the standard curve line, X=log starting copy number,b=Y-intercept or threshold fluorescence value.

Example 3 Construction of Recombinant Baculoviruses

Recombinant baculoviruses containing full-length (Ac_(—)1-787) anddeletion mutants (Ac_(—)1-415 and Ac_(—)416-787) of the HAstV-1(Newcastle) coat protein gene were generated with the BacPAK baculovirusexpression system kit (Clontech). To this end, the DNA fragment encodingthe cDNA of the coat protein (kindly provided by Dr. M. J. Carter,University of Surrey, England) (Willcocks, 1994) was amplified by PCRwith Pfu polymerase (Stratagene) and primers harboring BamHI and XbaIrestriction sites at the 5′ and 3′ end of the PCR product, respectively.Primers were designed to amplify the entire capsid gene coding region(aa 1-787) or the gene segments corresponding to aa 1-415 and aa416-787. Each PCR product was then purified by agarose gelelectrophoresis and the Gene clean II kit (Qbiogene), digested withBamHI and XbaI, and separately ligated into a BamHI/XbaI-digestedtransfer vector pBacPAK9. Following transformation of JM109 competentcells (Promega), plasmid DNA was isolated from several clones for eachconstruct and the presence of the inserted DNA was determined bydiagnostic restriction endonuclease mapping. Positive clones harboringthe various coat protein gene constructs were then completely sequencedacross the inserted DNA using Big Dye Terminator Sequencing Kit v 3.1 inan automated sequencing instrument (Applied Biosystems).

Generation of the recombinant baculovirus was carried out according tothe manufacturer's protocols (Clontech). Briefly, transfer vectorpBacPAK9, containing the HAstV-1 coat protein constructs wereindividually mixed with Bsu36I-linearized BacPAK6 baculovirus DNA andtransfected into Sf21 cells. Three days post transfection, cellsupernatants were harvested and putative recombinant viruses wereisolated by plaquing the supernatants once on Sf21 cell monolayers.Individual plaque isolates were amplified and titered followingconfirmation of the presence and expression of the inserted gene byimmunoblot analysis of the infected cell lysates (Dong X F et al., 1998.J. Virol. 72, 6024-6033) using a rabbit polyclonal antibody to HAstV-1particles (kindly provided by Dr. D. M. Bass, Stanford University, USA)(D. M. Bass and U. Upadhyayula. 1997. J. Virol. 71, 8666-8671) and ahorseradish peroxidase-conjugated goat anti-rabbit IgG secondaryantibody (Pierce). Signal detection by enhanced chemiluminescence wasperformed on a Versadoc instrument (Bio-Rad).

Example 4 Recombinant Protein Synthesis and Isolation

Sf21 cells (2×10⁸) in a 50 ml conical vial were infected with therecombinant baculoviruses at a MOI of 5 per cell. After 1 h at roomtemperature with rocking, the infected cells were transferred to aspinner flask containing cell growth medium plus antibiotics. Thespinner flasks were then allowed to stir at 27° C. Five days postinfection, cells were pelleted in a low speed spin and the medium wasdiscarded. Cell pellets were then frozen at −20° C. until needed.

The following protocol was developed to purify soluble coat protein fromAc_(—)1-787 and Ac_(—)1-415 infected cells. Unless otherwise indicated,all the following steps were carried out at 4° C. with pre-chilledbuffers and protease inhibitors (B.D. Pharmingen). Six frozen pelletswere each resuspended in 2 volumes of TNE (50 mM Tris [pH 7.0], 0.1 MNaCl, 10 mM EDTA) buffer and lysed by 3 cycles of freeze/thaw. Lysateswere centrifuged for 10 min at 13,300×g, and the supernatant wasdiscarded. The pellets, which contain aggregates of the coat protein,were each resuspended in 1 ml of TNE buffer containing 2% NP-40 andincubated on ice for 30 min. The resulting suspension was centrifuged at13,300×g for 5 min, and the supernatant was discarded. Each pellet wasresuspended in 1 ml of TNM (50 mM Tris [pH 7.0], 0.1 M NaCl, 20 mMMgSO₄) buffer plus 2 μl of 10 mg/ml DNase1 (Sigma), incubated 30 min atroom temperature and centrifuged for 5 min at 13,000×g, after which thesupernatant was discarded. The pellets were each resuspended in 1 ml ofTNE buffer using a pipette tip. The individual aliquots were pooled into2 tubes at this point and pelleted through a 1 ml 30% (wt/vol) sucrosecushion in TNE buffer at 234,000×g in a SW50.1Ti rotor (Beckman) for 1 hat 4° C. The supernatant was discarded and the pellet was thenresuspended with a syringe and needle in 1 ml of dissociation buffer(100 mM Tris [pH 9.0], 0.5 M NaCl, 100 mM urea, 10 mM EDTA, 5 mM DTT)and frozen overnight at −20° C. The next day, the solubilized proteinwas centrifuged at 13,300×g for 10 min and 500 μl of the supernatant wasloaded onto two 5-25% (wt/vol) sucrose gradient made in dissociationbuffer lacking protease inhibitors and spun at 274,000×g in a SW41Tirotor (Beckman) for 16 h at 4° C. After centrifugation, the coat proteinwas harvested by fractionation on an ISCO gradient fractionator at 0.75ml/min and 0.5 min/fraction. Fractions containing coat protein(typically fractions 6-12) were pooled and dialyzed against 100 mM Tris[pH 7.0], 500 mM NaCl, 10 mM EDTA overnight at 4° C. Samples were thenconcentrated (Amicon), aliquoted and stored at −80° C.

A different method was developed to purify soluble coat protein fromAc_(—)416-787 infected cells. Unlike Ac_(—)1-787 and Ac_(—)1-415,recombinant protein from Ac_(—)416-787 cells was soluble and could notbe purified as above. To this end, frozen pellets of Ac_(—)416-787infected cells were resuspended in 2 volumes of TNE buffer and lysed by3 cycles of freeze/thaw. Lysates were centrifuged for 10 min at13,300×g, and the supernatant was collected. Aliquots of the supernatantwere run on a 12% SDS-PAGE gel and the band corresponding to therecombinant protein was excised and eluted from the gel into TNE buffer.The protein was then stored at 4° C.

Example 5 HAstV-1 Coat Protein Displays Potent Complement Activity

The initial experiment that demonstrated coat protein activity oncomplement is shown in FIG. 2. In this assay, red blood cells (RBCs) aresensitized with antibody and incubated with normal human serum (NHS)causing lysis by complement activation (FIG. 2, NHS column). BSA (bovineserum albumin) is a negative control protein without complement effectsand shows an equivalent amount of RBC lysis compared to NHS alone (FIG.2, BSA+NHS column). Cobra venom factor (CVF) is a powerful activator ofcomplement that causes depletion of complement components, thusinhibiting lysis of the RBCs; here CVF is used as a positive control(FIG. 2, CVF+NHS column). When increasing amounts of HAstV-1 coatprotein was added to NHS, a dose response in RBC lysis was demonstratedindicating decreased complement activity. 1 ug of coat protein had anegligible effect whereas 8 ug exhibited >75% decreased RBC lysis by NHS(FIG. 2, last 4 columns on the right of graph). The fact that the coatprotein inhibited RBC lysis to a similar degree as CVF is striking giventhat CVF is considered the most potent complement-depleting compoundfound in nature. Similar results are shown in FIG. 10, which illustratesan independent set of experiments (mean values for each are presented,n=4) using modified amounts of HAstV-1 coat protein from 12.2 μg to 19.4μg added to NHS in the experimental samples. BSA and CVF controls,likewise, gave virtually identical results to those in FIG. 2.

Example 6 HAstV-1 Virions Also Display Potent Complement Activity

While the HAstV-1 coat protein had complement activity, additionalexperiments were conducted to determine whether authentic, infectiousHAstV-1 virions also strongly affected complement activity. To this end,varying amounts of virus in cell culture medium were added to NHS (FIG.3). As expected, NHS alone completely lysed the RBCs, whereas CVFblocked lysis strongly. As more virus was added to NHS, a dose responsewas seen with 70 ul of the virus inhibiting lysis to the same extent asCVF. At 85 and 100 ul of virus, inhibition of RBC lysis surpassed thatof CVF. As a control, NHS was incubated with equivalent amounts ofuninfected tissue culture medium to show that this activity was virusspecific (FIG. 4). Thus, both coat protein and whole virus demonstratedpotent effects on the complement system. The data in FIG. 3 and FIG. 4are presented side-by-side in FIG. 5, with viral genome copy number(×10⁸) added to the axis.

Example 7 HAstV-1 Virions and CVF Display Similar Kinetics in the RBCLysis Assay

To characterize the kinetics of activity of the astrovirus virions inthe RBC lysis assay, a time course comparing HAstV-1 virions and CVF wasconducted. HAstV-1 particles and CVF were incubated from 0 to 60 minutesin the presence of 2% NHS. At 0, 5, 15, 30 and 60 minutes, aliquots wereremoved and exposed to the sensitized RBCs (mean values are presented,n=3 to 5 for each sample). As shown in FIG. 6, both CVF and the virusshowed similar kinetics in the inhibition of RBC lysis. The 60 minutetime point suggests that the virus may have a stronger effect than CVFat this concentration as suggested above in FIG. 3 and FIG. 5. Whilethis assay does not identify the complement factor(s) with which thecoat protein/virus may interact, the data suggests that the coatprotein/virus may activate and possibly deplete complement components ina similar fashion to CVF. Alternatively, the coat protein or virus mayinhibit the formation of downstream complement complexes necessary forlysis of target cells.

Example 8 HAstV-1 Virions Affect the Classical and Alternative Pathways

Three pathways exist for the complement system, the classical (primarilyactivated by antigen:antibody interactions, mannan-binding lectin (bindsspecific polysaccharides on pathogen surfaces) and alternative (neitherantibody nor lectin dependent) pathways (FIG. 1). As depicted in FIG. 7,the astrovirus virions specifically interact with the classical pathwayleading to diminished activity as measured using Factor B depleted serain the RBC lysis assay. Factor B is essential for alternative pathwayactivation allowing specific testing of classical pathway activation(FIG. 7, factor B depleted sera column). As with CVF, astrovirusparticles inhibited RBC lysis in the absence of factor B indicating thatthis virus specifically blocks or depletes the classical pathway (FIG.7). Similar results were obtained with HAstV-1 coat protein asillustrated in FIG. 8 (mean values presented, n=4). Taken together,these results indicate that HAstV-1 coat protein suppresses serumcomplement more effectively using factor B-depleted serum versus NHS,suggesting that HAstV-1 coat protein acts more strongly on the classicalpathway than the alternative pathway. It is possible that the lectinpathway may also activate under these conditions.

While HAstV-1 coat protein has dramatic ramifications for the classicalpathway, HAstV-1 virions affect the alternative pathway to a lesserextent. As demonstrated in FIG. 9, in an assay to test for alternativepathway activation, NHS lyses rabbit RBCs as expected (NHS column). WhenCVF is added to NHS, lysis is significantly diminished (NHS+CVF column).In the presence of virus, lysis of cells is modestly affected (NHS+viruscolumn). As a negative control, NHS in the presence of cell culturemedium did not affect lysis as expected (NHS+media column). While thisassay is set up to detect alternative pathway activation (i.e., the RBCsare not sensitized with antibody), the presence of astrovirus antibodiesin NHS could potentially activate the classical pathway. To confirm thatthese findings were specific to the alternative pathway, C2 depletedserum (C2D) was utilized in the place of NHS (FIG. 9). By depleting NHSof C2 in this assay, the classical pathway and lectin pathways areblocked and any RBC lysis is due exclusively to alternative pathwayactivity. As expected, C2D in the absence or presence of cell culturemedium lysed RBCs (C2D and C2D+media column). Both CVF and virus showedsimilar levels of inhibition of RBC lysis in this assay. These resultsdemonstrate that HAstV-1 virions inhibit the alternative pathway to alesser extent then the classical pathway and it is possible that theviral coat protein functions to block both pathways by completelydifferent mechanisms. To further test for alternative pathwayutilization, C2D sera was incubated with rabbit RBCs alone inMg-EGTA-GVBS buffer or with 89 μg of purified HAstV-1 coat protein (n=3for each). The results, depicted in FIG. 10, demonstrate that whileHAstV-1 coat protein does in fact inhibit the alternative complementpathway as with virus (FIG. 9), the effect is minimal in comparison tothe inhibitory effect on the classical pathway as illustrated above inFIG. 7 and FIG. 8. Again, the data demonstrates that while somealternative pathway inhibition is observable, the effect of astrovirusvirions and coat protein is more significantly inhibitory of theclassical pathway than the alternative pathway.

Example 9 Overlay Blot Assay

To ascertain whether HAstV-1 coat protein binds to specific complementfactors, we utilized a modified virus overlay protein binding assay(VOPBA) approach (Borrow and Oldstone, 1992). To this end, 1 or 3 μg ofpurified complement factors C1, C1q, C1r, C1s, C2, C3 and C4 (Comptech)were mixed with 1×PBS and an equal volume of 2×SDS sample buffer lackingreducing agents and loaded onto a 7.5% SDS-PAGE gel. Followingelectrophoresis at 150V for 1 h, proteins were transferred tonitrocellulose and blocked in 5% NFDM-PBS-0.001% Tween-20 for 1 h atroom temperature. Purified WT coat protein was then added (−10-20 μg) tothe blot and allowed to incubate for 1 h, after which the blot waswashed extensively with PBS-0.001% Tween-20. The rest of the procedurewas carried out as for a standard immunoblot as described above.

An approximately 59 kDa band was detected in the C1 preparation. It iscontemplated that other preparations of complement proteins may interactwith wildtype astrovirus coat protein or derivatives under conditionsdiffering from those exemplified here. In order to further investigatethe binding of HAstV-1 coat protein to complement protein C1, theblotting protocol was repeated with purified C1 complex components C1q,C1r, and C1s. Total protein staining with Coomassie blue is depicted inFIG. 11A, while results for the blotting experiment are depicted in FIG.11B. The result suggests that HAstV-1 wildtype coat protein interactswith a C1q band in the 50-60 kDa range, and the C1r component binds justabove 80 kDa. However, subsequent experiments demonstrated that the 80kDa band was an artifact caused by the primary antibody to HAstV-1virions non-specifically interacting with the C1r homodimer in theabsence of the coat protein probe. As the data in FIG. 12C indicates(see below), the coat protein beinds specifically to C1q.

Further experiments confirmed the initial finding that HAstV-1 coatprotein binds C1q in an overlay blot assay. In FIG. 12A, purifiedcomplement factors C1, C2, C3 and C4 (Comptech) were mixed with 1×PBSand an equal volume of 2×SDS sample buffer lacking reducing agents andloaded onto a 7.5% SDS-PAGE gel without boiling the samples. Followingelectrophoresis at 150V for 1 h, proteins were transferred tonitrocellulose and blocked in 5% NFDM-PBS-0.01% Tween-20 for 1 h at roomtemperature. Purified coat protein was then added (˜10 μg) to the blotand allowed to incubate for 1 h, after which the blot was washedextensively with PBS-0.001% Tween-20. The rest of the procedure wascarried out as for a standard immunoblot using primary antibody toHAstV-1 virions and an appropriate HRP-conjugated secondary antibody. InFIG. 12B, the same experiment was performed as in FIG. 12A above, exceptno coat protein probe was utilized. In FIG. 12C, 3 μg of BSA, C1, C1q,C1r and C1s were mixed with 1×PBS and an equal volume of 2×SDS samplebuffer containing reducing agents, boiled and loaded onto a 12% SDS-PAGEgel. The overlay blotting was then carried out as in FIG. 12A. In FIG.12D, the overlay blot in FIG. 12C was stripped with Restore Western blotstripping buffer (Pierce) according to the manufacturer's guidelines andprobed with polyclonal antibodies to C1q, C1r and C1s (Santa Cruz)followed by the appropriate HRP-conjugated secondary antibody. FIG. 12Erepresents a duplicate blot as in FIG. 12C except no coat protein probewas utilized.

The above experiments demonstrate that the coat protein binds tosomething in C1 that migrates at about 59 kDa. Because these sampleswere not boiled or reduced and the C1 preparation purchased fromComptech is contaminated with other serum proteins, further experimentswere necessary to rule out the possibility that the coat protein wasbinding to something other than a C1 constituent, e.g., C1q, C1r, orC1s. The three highly purified C1 components, boiled and reduced, wererun on a gel and the experiment was repeated. This time, anapproximately 34-kDa band was detected in both the C1 and C1q lane. A34-kDa band is consistent with the C chain of the C1q protein. C1q, whenfully oxidized and reduced, breaks into three separate chains: chain Aruns at 27.5 kDa, chain B runs at 31.6 kDa, and chain C runs at 34 kDa(Cooper, N. R., 1985. Adv. Immunol. 37, 151-216). The overlay blot wasstripped and probed with antibody to C1q, C1r, and C1s. When theprevious blot was aligned to the re-probed blot, the band in the overlaycorresponded to the C1q C chain. The 59 kDa band seen in FIG. 12Aprobably represents one of the doublet bands seen in the C1q lane inFIG. 12D. This doublet is most likely a C-C chain and A-B chain dimer,which has been reported (Cooper, N. R., 1985. Adv. Immunol. 37, 151-216)to run at 54 kDa and 69 kDa, respectively. The sum of these experimentsis that astrovirus coat protein interacts with a C1q chain in this invitro binding assay.

Example 10 Other HAstV Serotypes Suppress Hemolytic Complement Activity

To determine whether HAstV serotypes other than the type 1 exhibit thesame effects on complement activity, equivalent amounts cell lysatesinfected with serotypes 1-4 (2.92×10⁸ genome copies each) were analyzedin the RBC lysis assay with all four serotypes demonstrating comparablelevels of hemolysis inhibition (Table 2). These findings suggest thatthe complement suppressing effect reported here is a conserved propertyof the HAstVs.

TABLE 2 RBC lysis assay on HAstV serotypes 1-4 HAstV serotype^(a)Inhibition of hemolysis (%)^(b) SE 1 86.3 2.38 2 84.3 3.14 3 86.3 3.14 487.0 0.47 ^(a)2.92 × 10⁸ genome copies from infected cell lysates wereutilized for each serotype tested. ^(b)n = 3

Example 11 Analysis of Recombinant Coat Protein Oligomerization

SDS-PAGE and immunoblot analysis of the HAstV-1 coat proteinpurification procedure and demonstration of spontaneous oligomerizationby the coat protein. As illustrated in FIG. 13A, aliquots of the first18 fractions from the sucrose gradient ultracentrifugation step of thepurification procedure were analyzed on 7.5% SDS-PAGE gels. The gelswere then stained with Coomassie blue. Fraction numbers are located atthe top of the gels and the arrows indicate the direction ofsedimentation from the top to bottom of the gradient. The migration ofthe 87 kDa coat protein (CP) is indicated. In FIG. 13B, immunoblotanalysis of the same gradient fractions utilizing an antibody to HAstV-1virions. FIG. 13C illustrates a 7.5% SDS-PAGE analysis of the coatprotein containing fraction at each stage of the purification procedureas analyzed by Coomassie blue staining and FIG. 13D represents theimmunoblot of the same gel. In FIG. 13E, aliquots of sucrose-purifiedcoat protein were either boiled or not boiled in the presence of2-mercaptoethanol and run on a 7.5% SDS-PAGE gel. The gel was thenstained with Coomassie blue. The boiled protein migrates atapproximately 87 kDa, the expected mass of the uncleaved coat proteinprecursor (monomer, M) whereas the unboiled sample migrates above 250kDa possibly representing a trimer (T) which would be postulated to runat 261 kDa. For all gels and blots, the molecular weight markers (inkDa) are indicated.

Example 12 HAstV-1 Coat Protein Inhibits iC3b Formation and Activationof the Terminal Pathway

Antibody sensitized sheep RBCs were incubated with NHS alone, with 1 μgCVF or with 76 μg of purified HAstV-1 coat protein (CP) for 3 hours at37° C. As represented in FIG. 14A, aliquots of each reaction were boiledand reduced, run on a SDS-PAGE gel, transferred to nitrocellulose andprobed with a polyclonal antibody to C3. Positive controls for C3 alpha(114 kDa) and beta (75 kDa) chain along with the two iC3b products (65kDa and 42 kDa) are indicated. These results demonstrate that NHS in thepresence of HAstV-1 coat protein does not generate significant amountsof iC3b. The presence of iC3b is an indication of C3 convertaseformation, i.e. activation of either the classical, alternative ormannose-binding lectin pathways. In FIG. 14B, an iC3b ELISA wasperformed on the samples using a monoclonal antibody to iC3b. Theabsorbance of the supernatants was read in a spectrophotometer at 405nm. A standard curve was utilized to determine the values of iC3b(ng/ml). Data are means of four independent experiments. Error barsdenote standard errors of the means. These results further confirm thatNHS in the presence of coat protein does not generate significantamounts of iC3b. The observation that even less iC3b is generated withthe addition of coat protein than with the addition of NHS alone isremarkable as NHS normally produces iC3b spontaneously, a process knownas “tickover.” Aliquots of the same 3 samples were subject to a SC5b-9ELISA using a monoclonal antibody to SC5b-9. The absorbance of thesupernatants was read in a spectrophotometer at 405 nm. A standard curvewas utilized to determine the values of SC5b-9 (ng/ml), as illustratedin FIG. 14C. Data are means of four independent experiments. Error barsdenote standard errors of the means. The ELISA data in FIG. 14Cdemonstrates that the terminal complement complex (known alternativelyas the membrane attack complex or “MAC”) is completely inhibited fromformation in the presence of astrovirus coat protein. These observationstaken as a whole indicate that astrovirus coat protein inhibits thecomplement system, a distinct mechanism from complement depletion asmediated by CVF. The fact that coat protein can inhibit the systemupstream of C3 is significant as this prevents the formation of the C3aand C5a anaphylatoxins as well as MAC formation. Prevention of thoseproducts of the complement cascade greatly enhance the therapeutic valueof the coat proteins because they fail to activate the cascade, unlikeCVF.

Example 13 Exogenous C1 Reverses the Inhibition of Hemolytic Activityand Deposition of C3 on Zymosan by HAstV-1

In order to further define the role of C1 in the inhibition ofcomplement-mediated lysis by HAstV-1, HAstV-1 inhibition of hemolyticactivity was tested for reversibility by the addition of exogenous C1.NHS was incubated with 47 μg coat protein (CP) or 1 μg CVF for 1 hour at37° C. 47 μg of CP was used to achieve approximately 50% RBC lysis.After the incubation, 10 μl of C1 (−1 mg/ml) or 10 μl BSA (1 mg/ml) wasadded back to the indicated samples as shown in FIG. 15A.Simultaneously, RBCs were added to all samples. Heme lysis wasstandardized to 100% for NHS alone. “HI-NHS” indicates the use heatinactivated NHS. Data from the RBC lysis assay are means of results fromfour independent experiments. Error bars denote standard errors of themeans. In FIG. 15B, 20 μl of NHS was added to either (i) 50 μl GVBS−−buffer and incubated alone, (ii) with 67 μg (50 μl) coat protein, orwith 1 μg CVF in 49 μl of GVBS++ buffer. All volumes were brought up to1 ml using GVBS++ buffer and incubated for 1 hour at 37° C. Afterincubation, 15 μl C1 (˜1 mg/ml) was added to the indicated samples andsubsequently 25 μl zymosan was added to all samples. After a 10 minuteincubation at 37° C., the samples were washed twice in GVBS++ buffer andtreated with 30 μl of 25 mM methylamine for 1 hour in a 37° C. waterbath before being spun down and the supernatant collected. A C3 ELISAwas performed on the samples using a polyclonal antibody to C3. Theabsorbance of the supernatants was read in a spectrophotometer at 405nm. A standard curve was utilized to determine the values of C3 (ng/ml).Data is presented as the mean values of four independent experiments.Error bars denote standard errors of the means.

In FIG. 15A, the amount of HAstV-1 coat protein was titered to an amountnecessary to achieve 50% RBC lysis. When C1 protein was added back tothe mixture, the hemolytic activity was completely restored. Incontrast, restoration of hemolytic activity by adding back BSA, CVF, orheat-inactivated NHS does not occur. In FIG. 15B, the C1 add-back datapresented in FIG. 15A was confirmed using a different approach. In thisexperiment, NHS is added to zymosan, which activates serum complementand leads to the deposition of C3 on the zymosan. Methylamine is thenused to strip the C3 off the zymogen and the C3 levels are assayed byELISA. In the presence of the coat protein, there is very little C3deposition, as expected. When C1 is added back, more C3 is present,indicating that the effect of the coat protein is overcome. Adding C1 toCVF, conversely, has no significant effect. The sum of the experimentsillustrated in FIGS. 15A and 15B indicate that HAstV-1 coat proteininhibits the classical pathway of complement activation throughinteraction with the C1 complex.

1. A method for inhibiting complement-mediated tissue damage comprising administering astrovirus coat protein or derivatives thereof.
 2. The method of claim 1, wherein the tissue damage is related to rheumatoid arthritis.
 3. The method of claim 1, wherein the tissue damage is related to systemic lupus erythematosus.
 4. The method of claim 1, wherein the tissue damage is related to multiple sclerosis.
 5. The method of claim 1, wherein the tissue damage is related to myasthenia gravis.
 6. The method of claim
 1. wherein the tissue damage is related to autoimmune hemolytic anemia.
 7. The method of claim 1, wherein the tissue damage is related to membranoproliferative glomerulonephritis.
 8. The method of claim 1, wherein the tissue damage is related to serum sickness.
 9. The method of claim 1, wherein the tissue damage is related to turkey astrovirus infection.
 10. The method of claim 1, wherein the tissue damage is related to Adult Respiratory Distress Syndrome.
 11. The method of claim 1, wherein the tissue damage is related to ischemia reperfusion Injury
 12. The method of claim 11, wherein the ischemia-reperfusion injury results from stroke.
 13. The method of claim 11, wherein the ischemia-reperfusion injury results from myocardial infarction.
 14. The method of claim 1, wherein the tissue damage is related to allo- or xeno-transplantation.
 15. The method of claim 14, wherein the tissue injury is further the result of hyperacute rejection.
 16. The method of claim 14, wherein the tissue injury is further the result of graft versus host disease (GVHD).
 17. The method of claim 1, wherein the tissue damage is related to Alzheimer's Disease.
 18. The method of claim 1, wherein the tissue damage is related to burn injuries.
 19. The method of claim 1, wherein the tissue damage is related to hemodialysis damage.
 20. The method of claim 1, wherein the tissue damage is related to cardiopulmonary bypass damage.
 21. The method of claim 1, wherein the tissue damage is related to Paroxysmal Nocturnal Hemoglobinuria. 